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Volume 8, Issue 5, 2018, 3635 - 3643 ISSN 2069-5837

Biointerface Research in Applied Chemistry

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Review Article Open Access Journal

Received: 09.09.2018 / Revised: 05.10.2018 / Accepted: 10.10.2018 / Published on-line: 15.10.2018

Leukocyte Platelet-Rich Fibrin (L-PRF), a new biomembrane useful in tissue repair: basic
science and literature review

Alessandro Crisci 1, 2, 3, *, Giulio Benincasa 4, Michela Crisci 5, Francesco Crisci 1

1
Unit of Dermosurgery Cutaneous Transplantations and Hard-to-Heal Wound, “Villa Fiorita” Private Hospital, 81031 Aversa CE, Italy
2
School of Medicine, University of Salerno Italy, 84084 Fisciano SA, Italy
3
Institute for the Studies and Care of Diabetics, Abetaia, 81020 Casagiove CE, Italy
4
Pathological Anatomy, ”Pineta Grande” Private Hospital, 81030 Castelvolturno CE, Italy
5
Faculty of Medicine and Surgery, Vasile Goldis Western University of Arad, 310025 Arad, Romania
*corresponding author e-mail address: alessandrocrisci@libero.it

ABSTRACT
PRF is a second-generation platelet concentrate and can be distinguished in P-PRF (Pure Platelet-rich fibrin) and L-PRF (Leukocyte and
Platelet-rich fibrin). In vivo, during the platelet clot formation, platelets are bound to β-integrin fibrin and the clot retracts itself. The PRF
clot forms a solid fibrin matrix, displaying a complex tridimensional architecture. Measuring the levels of PDGF-BB, TGF-1β and IGF-
1, in the PRF matrix, it was revealed that slow fibrin polymerization during PRF elaboration leads to the intrinsic platelet formation of
cytokines and glycan chains inside the fibrin mesh. The PRF proximal part (the part closer to the red clot) contains platelets and
leukocytes, found to be massively enclosed inside the fibrin net. Platelet localization inside PRF was examined through immunostaining
and scanning electron microscope (SEM). Compared to the abrupt basal compression of PRF membrane (G-PRF), preservation of
plasma level, of 3D fibrin, and of platelets was found to be more intact in PRF membrane preparations obtained through metallic
compression system (PRF-C) (L-PRF Wound Box). The pure platelet-rich fibrin (P-PRF) and the leukocyte and platelet-rich fibrin (PRF-
L) are biomaterials with solid fibrin or biomaterials containing leukocytes. No platelet concentration related problems were found in
PRF, as all platelets in the drawn blood sample are activated and integrated inside the fibrin matrix of the clot. Approximately 97% of
platelets and more than 50% of leukocytes are concentrated inside the PRF clot, which displays a specific tridimensional distribution.
Almost all platelets (>97%) are absent inside test-tubes of groups tested after PRF membrane extraction.
Factors freed by platelets contained in L-PRF induce and control the proliferation and migration of other cell types, involved in tissue
repair, like smooth cell muscles (SMCs) and mesenchymal stem cells (MSCs).
Keywords: blood derivatives, growth factors, leucocyte and platelets rich fibrin, L-PRF wound box, stem cells.

1. INTRODUCTION
Recent scientific evidence suggests that platelets could play
a new role in tissue repair and in vascular remodeling, other than
being active actors in inflammatory and immune responses.
Platelets release biologically active proteins and other substances,
able to influence a series of processes, favoring cell intake, growth
and morphogenesis. These substances are released or exposed on
the surface of activated platelets. The platelet capability of
releasing substances inside a clot makes the clot itself a natural
autologous source of growth factors and cytokines, which can be
therapeutically used to accelerate and speed up physiological
healing processes. Many of these substances are gathered and
stored in platelets α-granules, easily identified with Scanning
electron microscope (SEM) and with immunofluorescence
staining.
Thin fibers contained in HPC (Human Platelet Concentrate)
could be related to the high initial concentration of platelets in
HPC (3-5 x 1011 platelets/l), where local procoagulant activity
could even be improved through the start of pro-thrombotic
stimuli amplification, and it leads to an almost explosive thrombin
production, thus causing an increase in fibrinogenesis on the
surface of platelets, which in turn leads to fibrin formation and
polymerization [1].
Adhesive proteins are rather abundant on the fibrin
reticulum: Fibrinogen (Fg), Fibronectin (Fn), Vitronectin (Vn),
Thrombospondin-1 (TSP-1). Fn concurs to wound healing and
promotes the mitogenic activity of Platelet Derived Growth Factor
(PDGF). Among the platelet-stored Growth factors (GF), essential
for wound healing, we count PDGF, in particular, PDGF–AB and
PDGF–C (prevalent isoforms in platelets); other factors include
the Vascular-endothelial growth factor (VEGF), essentially
VEGF-A, Transforming growth factor β1(TGF-β1), basic
Fibroblast growth factor (bFGF), of the FGF-2 family; Epidermal
growth factor (EGF), Hepatocyte growth factor (HGF) and
Insulin-like growth factor-1(IGF-1). Members of TGF-β family
are prominent in wound healing and scar formation. Holding a
preponderant role in healing, platelets are a rich source of
cytokines and chemokines. An example is RANTES, a chemokine
deposited on the inflamed endothelium thanks to a P-selectin-
platelet-dependent mechanism.
In general, these factors chemotactically recruit and
activate stem cells, as well as induce their mitogenesis and
differentiation. Platelets are also a potential source of matrix
metalloproteinases (MMPs) (MMP-2, MMP-9, ADAM-10,
ADAM-17, ADAMTS-13), as well as tissue inhibitors of

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 Alessandro Crisci, Giulio Benincasa, Michela Crisci, Francesco Crisci
metalloproteinases (TIMP 1-4). MMPs are found in α-granules rich plasma); L-PRP (Leukocyte and Platelet-rich plasma); P-PRF (Pure
and in cytoplasmic membrane vesicles as well. Platelet-rich fibrin); L-PRF (Leukocyte and Platelet-rich fibrin).
The PRF clot is created by a natural polymerization
Fibrinogen could improve wound scarring, increasing both
process, during centrifugation, and its natural fibrin architecture
cell proliferation and migration; it assembles with Fn in fibrils,
seems to be responsible for a slow GF and matrix glycoproteins
without taking into account fibrin formation. Wound healing
release (≥7 days). PRF clots are directly used to fill cavities in
outcomes are influenced by fibrin structure (fiber thickness,
plastic and general surgery interventions. Even though platelets’
branching points number, clot porosity and permeability) where
GFs cover an essential role in PRF biology, fibrin architecture,
the wound is present [2].
leukocyte-contained substances and stem cell presence are three
It appears clear that platelet-rich fibrin clots can be
key parameters. Platelets and leukocytes distribution inside fibrin
considered a bioactive reservoir. An elevated hematocrit or a low
clots is highlighted through blood count, photon microscopy and
platelet count could be a limiting factor, and further research is
SEM analyses (Figure 2).
needed to establish an optimal platelet count for the material to be
used in procedures. Beside secreting proteins, platelets are able to
release low molecular weight diffusible compounds, and a great
number of microparticles, which carry proteins like TF or IL-1,
that is pro-thrombotic substances. Therefore, application in
patients displaying hereditary or acquired thrombotic risk factors
(including arterial hypertension, Factor V Leiden mutation).
Contemporary administration of anti-platelet drugs could
theoretically limit the efficacy of the treatment. Aspirin reduces
platelet secretion and its administration should therefore be
avoided in the days immediately preceding autologous L-PRF
preparation since it inhibits cyclooxygenase enzymes (COX).
PRF® (Platelet-rich fibrin) is a new generation platelet
concentrate, obtained through autologous peripheral blood
centrifugation, without any biological agent addition. It includes,
besides platelets, the fibrin matrix polymer, leukocytes, cytokines
and stem cells. It is distinguished in P-PRF (Pure Platelet-rich Figure.2. SEM picture displaying: 1 the fibrin-rich layer (5.000 x
fibrin) and L-PRF (Leukocyte and platelet-rich fibrin) (Figure 1). magnification); 2 a zone of enriched platelets with various degree of
activation (.1.500 x magnification); 3 buffy coat with numerous
Another platelet concentrate, PRP, has a transitory effect on
leukocytes and 4 the red blood cell base (2.000 x magnification).
wound healing; furthermore, bovine-derived thrombin employed
in its formation increases the coagulopathy risk, not seen with An adequate PRF® preparation method should properly
PRF® instead. With its (PRF®) use, a natural coagulation process separate platelets from erythrocytes and concentrate them without
takes place, allowing for an easy leukocyte and PRF® (L-PRF®) damaging or lysing platelets themselves. Growth factors contained
collection inside the clot. inside α-granules are not active during secretion, but get activated
Fibrin gels are recommended as scaffolds, to be employed through fusion with platelet membrane. Consequently, if platelets
in tissue engineering in many respects. The most prominent reason are damaged during PRF production, they will not secrete
is its compatibility with fibrin cell life, which differs based on its bioactive growth factors anymore. Platelets are, as a matter of fact,
various and numerous components and the processes involved in particularly labile, and sensible to every kind of stressful event
scaffold formation. In vivo, at the end of the fibrin clot formation, during their processing and application; for this very same reason,
platelets bind the fibrin to β-integrin and the clot contracts. The GFs concentration could be influenced by handling during blood
clot contracts also along the wound margins, giving rise to tension processing events. It is then decisive the type of centrifugation
forces and directing the new temporary matrix. applied, which needs to respect several demands and
characteristics, including an initial Low Start, a high-frequency
centrifugation as middle phase and a closing Low Stop [3], and it
must take place at a set temperature and for a specific time interval
(Crisci et al. 2015)[4, 5].
Here, we review the spectrum of platelet-rich blood
derivatives, discuss their current applications in tissue engineering
Figure.1 Different types of human platelet concentrates (HPCs): PRP and regenerative medicine, reflect on their effect on stem cells,
(Platelet-rich plasma); PRF (Platelet-rich fibrin); P-PRP (Pure Platelet- and highlight current translational challenges.

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 Leukocyte Platelet-Rich Fibrin (L-PRF), a new biomembrane useful in tissue repair: basic science and literature review
2. EXPERIMENTAL
2.1. L-PRF processing. PRF® production protocol is very simple:
blood is immediately centrifuged within 2 minutes from
withdrawal, following the subsequent steps: 30” of acceleration, 2’
at 2700 rpm, 4’ at 2400 rpm, 3’ at 3000 rpm, and 36” of
deceleration and arrest. The resultant product is made of three
layers: PPP (Platelet-poor plasma at the top), PRF (central clot),
Red Blood Cells (RBCs) at the bottom (Figure 3). Resulting PRF
clots are gathered and red blood cells are removed with the aid of
scissors, without macroscopical damage at PRF structure expense.
Figure 3. Different phases of PRF preparation (see text).
Fibrinogen is initially concentrated in the middle and
superior portion of the test tube, that is in between red blood cells
(RBCs) at the bottom and the Platelet-poor plasma (PPP) at the
top. Clot compression by means of a compression system (L-PRF
box) significantly stimulates cell proliferation and
neovascularization [6].
Quantifying the levels of PDGF-BB, TGF-β1 and IGF-1 in
PPP and in PRF, analyses showed that slow fibrin polymerization
during PRF processing leads to the intrinsic production of
cytokines and glycan chains by platelets inside the fibrin mesh.
Analyzing three pro-inflammatory cytokines (IL-1β, IL-6, TNF-
α), one of the anti-inflammatory cytokines (IL-4) and the
angiogenesis promoter (VEGF), we saw that PRF could be a
central junction in immune regulation, with the capability to
control inflammation. PRF, differently from other platelet
concentrates, could be able to progressively release cytokines
during fibrin matrix remodeling.
Platelet cytokines and leukocytes are an important factor in
determining the effects and role of this biomaterial, but, both the
fibrin matrix and its contained elements are responsible for PRF
therapeutical enhancement (Tab.1). Cytokines are immediately
used in wound healing. The mechanism involved in PRF
formation is the concentration of fibrinogen at the top of the test
tube, which combines with circulating thrombin, due to the
centrifugation process, essential in fibrin formation. PRF central
portions contain platelets, massively encased inside the fibrin
mesh. The therapeutical success of this kind of technique is
entirely dependent on the time interval between blood sample
withdrawal and its centrifugation, which should be carried on in
the shortest interval possible, as well as on processing temperature
and the type of test tube employed.
Blood samples should be withdrawn from patients that
have no previous history of aspirin or other anticoagulant drug
administration up to two weeks prior the procedure.
Dohan Ehrenfest et al.(2012) [7] detected a slower GF release in
PRF compared to PRP, and they observed improved healing
capabilities with the employment of PRF. Also, it was
demonstrated that cells are able to migrate inside the fibrin
network. A slow polymerization procedure imparts a physiological
architecture to the PRF® membrane, particularly favorable to
sustain healing processes.
In addition to standard formulations, PRF can also be
obtained in an injectable form (I-PRF). I-PRF is obtained by
producing a PRF membrane, which subsequently is compressed
between metallic sheets. Advantageously, this injectable material
can coagulate immediately post-injection to form a biomaterial as
well as be combined with any biomaterial of choice for non-
covalent incorporation [8].
2.2. PRF effects in tissue engineering. Platelet localization inside
the PRF gel was examined through immunostaining and with the
aid of Scanning Electron Microscope (Figures 2, 4) taken from
Kobayashi et al. 2012 [6].
Su and Burnouf [9] demonstrated that a copious amount of
growth factors was discarded when pressing took place. Hence,
pressing processes could influence the efficacy and clinical quality
of PRF preparations, to be used as graft material.
Platelet-derived mediators induce and regulate fibroblasts’
late action, and leukocytes’ recruitment, neutrophils first, followed
by macrophages, consequently eliminating dead cells and cellular
debris. Moreover, factors derived from platelets induce and
control proliferation and migration of other types of cells, which
are critically involved in tissue repair, like smooth muscle cells
(SMCs) and mesenchymal stem cells (MSCs).
Activated platelets release a whole range of chemokines
and promote adult stem cells’ absorption, adhesion and
proliferation, including progenitor CD-34 positive cells, MSCs,
SMC progenitors and endothelial progenitors. The multipotent
nature of these cells and their capability to increase vascular tissue
repair, due to paracrine mechanisms, makes them good candidates
as therapeutical vehicles to be employed in regenerative medicine
fields. Moreover, tissue damages themselves are able to generate
strong chemoattractant signals, affecting stem cells, and providing
their regenerative action basis.
Platelets regulate adult stem cells recruitment toward
damaged cells and could therefore, constitute an essential
mechanism for regenerative cellular processes. Activated platelets
release HGF and have been linked to MSCs passage through
endothelial cells, lining human arteries. Human mesenchymal
stem cells’ proliferation (hMSCs) is proportional to platelet
concentration inside PRF concentrates.
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 Alessandro Crisci, Giulio Benincasa, Michela Crisci, Francesco Crisci
Table 1. Leukocytes, RBC and Platelets number in whole blood (control group) and red clot after PRF membrane collecting (test group)
(from Crisci et al. 2015).
Leukocytes/µl RBC/µl
Mean Range Mean
Controll 6.900 6.100- 5.19 (106) 5.01-5.52 (106)
7.800
Group 1 3.500 3.000- 5.89 (106) 5.75-6.08 (106)
3.800
Group 2 3.600 3.300- 5.84 (106) 5.78-5.91 (106)
4.000
Figure 4. Microscopy picture at S.E.M. Hitachi Tabletop Microscope
TM3000 of L-PRF.
On the left: Fibrin mesh (600x magnification); on the right: activated
platelet (4000x magnification)
Among tested growth factors, PRF contained PDGF
constitutes the major portion, and stimulates, significantly, cell
proliferation and neovascularization. An important PRF
characteristic is the resulting fibrin gel, shown to be denser than
the gel prepared with thrombin addition (PRP).
Thus, the establishment of a standard protocol for PRF
preparation was necessary, satisfying the following criteria:
1) Platelet-contained growth factors should be preserved to
stimulate surrounding host cells;
2) Platelets should be stored inside the fibrin mesh with minimal
damage or activation;
3) The tridimensional fibrin mesh must be used as a scaffold by
surrounding host cells.
The PRF was subdivided into 3 regions, of equal length
(Fig.3) and platelet presence in each region was observed through
S.E.M., through Optic Microscope in horse-derived preparations
(Crisci e al.2017)[10-12].
Region 1 is the region closest to the red clot, and shows a
conspicuous number of platelets aggregates, displaying some
lymphocytes and other white blood cells. Platelet count is reduced
as the distance from the red clot is increased. Inside region 2
(central region), we observe fibrin fibers (primary and secondary
fibers) and some platelets. Inside region 3, the fibrin mesh is
extremely evident, while the platelet count is low (Figure 5).
We identified some anti-CD41 antibody positive cells,
through immunocytochemistry, and, as a matter of fact, in L-PRF,
on one side of the membrane, many CD41-positive platelets were
gathered, and some platelets could be found inside the membrane.
On the membrane’s opposite side, only a few platelets were
observed. The discovery explained in Kobayashi et al.[6] studies
are constituted by the fact that platelets are not equally distributed
inside and on the surface of the PRF clot, even if the clot is
considered as a gel with uniform platelet concentration. Therefore,
in a clinical setting where platelet-derived growth factors are
Platelets/µl
Range Mean Range
2.66 (105) 2.18-3.09 (105)
6.000 4.000-8000
7.000 6.000-9000
expected and desired, the red clot-adjacent region must be used,
being richer in platelets.
Basing our actions on the assumption that PRF-retained
serum could contain elevated GFs levels, released by platelets,
which are more or less active during centrifugation phases, we
didn’t try to squeeze all the plasma with a complete compression
of PRF clots. The C-PRF tendency to contain higher growth
factors levels (PRF compressed through metallic compression
system), compared to G-PRF (PRF compressed with gauze), could
be traced back to platelet-derived GFs (PDGF-AA, PDGF-AB,
PDGF-BB).
This obtained result could be due to fibrin, since the
fibrin mesh could directly absorb GFs or could entrap serum
albumin or heparin, hence indirectly retaining GFs. It is almost
impossible counting and regulating the platelet count in PRF
preparations before clinical usage. Therefore, the clinically most
effective protocol to check result quality is using the PRF region
closest to RBC clot.
Cell migration was performed through the employment of
MSCs (mesenchymal stem cells), derived from human bone
marrow, and human umbilical vein endothelial cells (HUVECs)
[13]. MSCs migrated principally at day 3 for L-PRF preparations.
A higher migration rate was observed for L-PRF compared to L-
PRP at day 3, day 7 and day 14. HUVECs migration also reached
its peak at day 3, day 7 and day 14 for PRF preparations.
2.3. Preclinical Studies. Fibrin is a useful substrate for
bioengineering purposes, and it is one of the most popular
hydrogels in tissue engineering and regenerative medicine.
Grafted cells require specific signals from the extracellular matrix
to survive.
L-PRF is a preparation that contains leukocytes, and with
a high density of the fibrin mesh. These products exist as activated
gels, and cannot be injected or used as traditional fibrin glue.
However, due to their strong fibrin matrix, they could be handled
as a solid material, for purposed applications, achieving interesting
results in reconstructive surgery; even so, these applications are
still in an experimental phase, since we still lack a necessary
protocol for the usage of clots in various, specific surgical
procedures.
Fibrin generates a temporary matrix at the graft site, but its fibers
have no direction or tension, and it has a low count of associated
growth factors. Fibrin gels, instead, show a regular, reticular
disposition of its pores, with short, thin fibers, non completely
acellular; through SEM, platelets and leukocytes were observed
(Figure 5).
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 Leukocyte Platelet-Rich Fibrin (L-PRF), a new biomembrane useful in tissue repair: basic science and literature review
Studies carried out on horse models showed that platelet-
rich fibrin gels (PRFG) display big, dense fibers, which are
casually oriented (with a mean diameter of 117.7±10.53 nm).
Fibrin gel fibers are smaller (56.8±5.11 nm). Platelet incorporation
inside the fibrin gel results in structural alterations and an
increased growth factors’ concentration, hence it could improve
cell grafts’ performances inside the scaffold after transplantation.
In accordance with their growth factor storage, platelet directly
contributes to tissue growth, development and repair. PDGF and
TGF-1 are the most abundant growth factors, contained inside
platelets’ -granules and they get released inside the extracellular
space, after platelet activation. These cell factors orchestrate
proliferation, cell differentiation, matrix production, angiogenesis,
and wound contraction; growth factors supplementation is able to
improve transplanted cell survival and differentiation, in a number
of materials and tissues treated [14].
Platelet addition to the fibrin gel (PRFG) is related to an
increase in fibers’ diameter and the reduction of porous areas; they Figure 5. The three C-PRF regions, and O.M. platelet distribution
also increase the fibrin gel rigidity. Measured fiber diameter could observation on membrane surface. C-PRF was subdivided in three
be associated with the lower platelet concentration (100 x 103 regions: Region 1 adjacent to the red clot (RBC), Region 2 is the central
part and Region 3 is the distal part from the red clot. Platelet distribution
platelets/l), which is approximately closer to systemic platelet was observed in region 1 (A-D-E), region 2 (B-F) and region 3 (C-G).
concentration under standard, normal conditions. One possible Platelets are at higher concentration in region 1 and at lower
explanation could be that a lower platelet count would cause concentration in region 3. (from Crisci A. et al. 2017) Membrane L-PRF
0 min after compression (hematoxylin-eosin staining). (A) III proximal
decreases in tension applied to fibers.
25× white blood cell-pattern fibrin; (B) medium-III 60× erythrocytes
Porous areas and porous percentage are important structural pattern fibrin; (C) III distal 60× pattern fibrin; (D) III proximal 25×
indices for every biological scaffold. Bigger pores favor internal erythrocytes-fibrin; (E) III proximal 60× fibrin on the right, the center
growth and cell proliferation, while smaller pores favor cell lymphocytes, erythrocytes, and granulocytes neutrophils on the left; (F)
average III 25× pattern of fibrin; (G) III distal 60× pattern fibrin; (H)
adhesion, thanks to a bigger surface. smear of red clot 40× presence of monocyte in a carpet of red cells; (I) red
Under a clinical point of view, L-PRF shows excellent clot smear 40× presence of red blood cells, monocytes and platelets; (J)
handling properties: single L-PRF clots can be turned into red clot smear 100× presence of platelets in a carpet of red cells (staining:
may-grǔnwald-giemsa). Reprinted from an open access source [11].
membranes of fitting thickness and dimensions, thanks to the new
“L-PRF Wound Box” (Figure 6); merging two or more membranes
is useful to create a bioactive membrane of bigger dimensions, to
cover and form bigger grafts. L-PRF membrane can also be cut
and tailored. Being flexible enough, it adapts to different
anatomical areas.
Legally, a physician, is authorized to perform
endovenous injections, is also authorized for blood withdrawal.
Furthermore, as stated by the European Community regulations, Figure 6. L-PRF Wound Box.
hence with a law judged valid throughout Europe, L-PRF is not
2.4. L-PRF in Vitro. In vitro, L-PRF membrane and P-PRP (Pure
considered a hematological derivate, since there are no added
Platelet-rich plasma)(PRFG-endorest) gel components were
substances, but it is included under autologous cell grafting.
compared, through the evaluation of the slow growth factors and
Since the wound healing process achieved with this technique is
matrix molecules’ release.
not by primary or secondary intention, it is defined as “wound
These two gel families were seeded on a growth medium
healing by modified secondary intention” [15].
for 7 days, and the slow versions of 3 key growth factors (TGF-β1,
L-PRF family adapts itself to the requirements of the
PDGF-AB, VEGF) and 3 coagulation proteins and 3 matrix
various surgical intervention. Just like clots and membranes, L-
proteins (TSP-1[Thrombospondin 1]), (Fn) e (Vitronectin) were
PRF has shape and volume easily combined with the great bulk of
experimentally quantified for seven times (at 20’, 1h, 4h, 24h,
surgical techniques, as a filling method and healing biomaterial
72h, 120h, 168h)(Figures 7, 8, 9)(Crisci A.et al.2015)[4-5].
apposition, or as protecting membranes for wound healing.
These studies showed that products display two very
Furthermore, it is easy to prepare, also in great quantities, and very
different profiles: L-PRF membrane stayed solid and intact after at
cheap, making it particularly apt for everyday clinical practice. It
least 7 days and releases continuously a big quantity of growth
was successfully used by AA, especially in the treatment of
factors, a significative portion of which is produced inside the
cutaneous diabetic ulcers, also with osteomyelitis presence [16].
membrane. On the contrary, clear P-PRP releases the major

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 Alessandro Crisci, Giulio Benincasa, Michela Crisci, Francesco Crisci
portion of growth factors within the first hours and it is completely significant GF quantity, and it is particularly obvious with PDGF-
dissolved after 3 days (Figures 7-9). AB, because this growth factor is only released by platelets.
Leukocytes found in L-PRF are not only inflammatory
cells, as they show anti-nocireceptor effects as well, through the
release of different chemokines, anti-inflammatory cytokines (IL-
4, IL-10, IL-13) and peptidic opioids β-endorphins, dimorphin-A
etc.), hence they could promote a clinically relevant inhibition of
pathological pain (centrifugation process could delicately activate,
or stimulate in a pathological way, the inflammatory state, or
destroy leukocytes altogether). Many other types of cell are
present in these preparations. The leukocyte count is an important
parameter: lymphocyte populations are very different and do not
have the same impact as monocytes and granulocytes do.
Furthermore, many other cells, like circulating stem cells, could be
found in a platelet concentrate in a non-unimportant manner [13].
A certain quantity of GFs was observed in the serum released by
PRF (PRFR) and in the supernatant serum (SS), right after PRF
formation. An additional GF release from PRF was found up to
300’ after preparation (5h). SS contained quantities are  7 ng/ml
Figure 7. TSP-1 and Fibronectin variations in time. L-PRF comparison
(PDGF-AB), 9.5 ng/ml (TGF-β1), 0.1 ng/ml (VEGF) and with PRGF (Plasma rich in growth factors = P-PRP).
constitute a good index of clot-contained GFs’ basal levels inside
PRFR. On the contrary, it does not influence other intrinsic
True PRF is always autologous and not homologous. One growth factors that are released slowly in elevated quantities for
example of this misconception is the use of lyophilized platelets several days. The released quantities of VEGF and TGF-β1 are
from donors. Homologous platelets cannot be used and cannot massively produced by leukocytes.
secrete bioactive GF. Homologous platelet is antigenic due to their A non-standardized blood withdrawal procedure, slow and
abundance of cell membranes. inadequate, leads to a small, PRF-like fibrin mass, with unstable
PRF and SS contain great quantities of GFs, and do not fibrin polymerization (with consequent poor mechanical
need to be discarded as they could be useful in patient treatment properties), and to an unknown, not-reproducible growth factor. In
[9]. The initial PRF exudate (rich of growth factors and serum addition, it is very difficult to divide these small fibrin masses at
proteins) is collected in the container and PRF membranes are the RBC base, resulting in a heavy red blood cells mass in the
stored in a serum-humidified environment. This is an effective product [9].
method from a biological point of view. A new device, that we
have tested in L-PRF clots and membranes preparation and
procedure standardization, is L-PRF Wound Box (Crisci A. et al.
2017)[10-12]. It is an instrument where PRF clots can be turned
into membranes. L-PRF clot contains almost all of the platelets
and more than 50% of white blood cells coming from the initially
drawn blood (Tab.1); furthermore, it shows a strong fibrin
architecture and a particular tridimensional distribution of platelets
and leukocytes. This device permits clot preservation in a humid,
sterile environment for 1 hour, and it allows an increase in total
growth factor release. Mean PDGF-AB produced quantities are
significantly higher in every experimental instant (Figure 8) and
TGF-β1 (Figure 8) and VEGF (Figure 9) are significantly higher
during the first 4 hours. Numerous studies have confirmed the
gradual release of PDGF and TGF for 28 days from PRF
formation [16]. A possible explanation is that PRF polymerizes
with a 3D structure progressively, slowly and naturally during
Figure 8. TGF-β1/PDGF-AB and Vitronectin variations in time. L-PRF
centrifugation, and this helps to entrap cytokines released by comparison with PRGF.
platelets with the fibrin polymer. In addition, using the PRF Box,
the clot compression into the membrane is carried out through a
light compression, slow and homogeneous, and the resulting
membrane always remains homogeneously wet and soaked by
serum. This delicate method avoids extraction and loss of a

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 Leukocyte Platelet-Rich Fibrin (L-PRF), a new biomembrane useful in tissue repair: basic science and literature review
Figure.9 VEGF variations in time. L-PRF comparison with PRGF
2.5. PRF limitations. Platelets, fibrin, and leukocytes naturally
act synergistically to promote wound healing and tissue
regeneration, and the amplification of this
coagulation/regeneration effect on a surgical site or wound is the
aim of surgically oriented platelet concentrate employment.
According to POSEIDO classification, all products of this
category are gathered under the general name of human platelet
concentrate (HPC), whichever may their form or cell content be.
In addition, it is important to highlight the key role of leukocytes
and their influence, as well as fibrin architecture, concurring to the
potential clinical or experimental effects of these products, and it
is also important to emphasize how each different product is
referring to a precise biological distinctive composition.
Limitations that were found in the clinical setting and usage of
these products include:
1) Since PRF is an autologous product, an increased
requirement for the biomaterial availability is difficultly achieved.
Therefore, its use in surgical procedures must be tightly
controlled.
2) PRF contains circulating immune cells, as well as
antigenic molecules that prevent its use as allogenic material; an
increased risk for the transmission of infectious diseases is also to
be taken into account.
At this point, among the different parameters that were
not included in this type of classification, we recognize platelet
concentration, leukocyte concentration and the proportional
amount of the different leukocyte types. Platelet concentration-
related problems are non-existent, as all platelets included in the
blood sample are activated and integrated inside the clot’s fibrin
matrix. Concerning the leukocytes’ count and concentration, their
influence should be studied with particular care, as their presence
or absence could explain the conflicting results we observed
(Tab.2) [11].
A significant correlation between platelet counts and
TGF-β1 (p=0.005) and PDGF-BB (P=0.04) was found. TGF-β1 is
quantitatively increased in slow release PRF, compared to fast
release PRF (Figures 7, 8). WBCs, entrapped inside the PRF
matrix, were shown to be the main source of this TGF-β1
supplemental release, and these WBCs keep secreting this GF
inside the PRF clot for several days.
PDGF-BB is almost exclusively contained inside
platelets’ α-granules, and is released when they are activated,
hence explaining why its production is at maximum levels in
activated PRP and PRF, while it is reduced in slow release forms.
It is possible that hematological levels cannot be used to forecast
the immediate or slow release of GF inside PRF compounds [14].
Dohan Ehrenfest et al.[18] studies aimed to determine cell
composition and tridimensional organization of this autologous
biomaterial, and the utility of different test tubes (dry glass, coated
glass, plastic), all of them without gel. Almost 97% of platelets
and more than 50% of leukocytes were concentrated inside the
PRF clot, demonstrating a specific tridimensional distribution.
Platelet and fibrin are present in big quantities inside the first few
millimeters of the membrane, past the RBCs region boundaries.
There is no discernible difference in PRF architecture using
different types of test tubes.
2.6. Platelets and leukocytes analysis. Almost all platelets
(>97%) were absent inside test tubes of the group analyzed after
the PRF membrane extraction. In the test groups, the leukocyte
level significantly dropped compared to the control group
(p<0.01) and more than half of the leukocytes seemed to have
disappeared (Tab.2).
Missing platelets and leukocytes are trapped inside PRF
matrix when we use the scissor based collection method. The
absence of differences between the two test groups (p>0,05) seems
to indicate that clot compression does not influence the possible
release of cell bodies trapped inside the fibrin matrix.
In test groups, the lymphocyte concentration is
significantly lower, while granulocyte concentration is
significantly higher (p<0.01) compared to control group (Tab.3).
This would signify that lymphocyte were trapped inside the PRF
matrix more than other types of leukocytes. Lastly, mean platelet
volume (MPV) is noticeably reduced between test groups and
control groups (p<0.01); it went down from m3 (range:8-11 m3)
in whole blood to 4.7m3 (range: 4,5-5,8 m3) in test groups. This
phenomenon could be due to plasma osmolarity increases inside
test tubes after coagulation cascade activation.
2.7. Blood elements distribution analysis. Histomorphometric
analysis was performed using an optical microscope with 100x
total magnification. A 10 mm eyepiece, with 100 divisions
reticulum, was used to measure the coverage percentage of the
total length with at least one cell layer, in each section.
With hematoxylin-eosin staining, the fibrin matrix
appears homogeneous, in a pinkish color, while platelet aggregates
are in deep blue/violet. RBCs and leukocytes’ cytoplasm are deep
pink and not easily distinguished. Leukocytes’ nucleus is stained
in blue with hematoxylin and it is not easily distinguished from
platelet aggregates. With Masson trichrome staining (modified by
Godman), platelet aggregates are still seen in deep blue, but RBCs
are easily distinguished since they are stained in red.
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 Alessandro Crisci, Giulio Benincasa, Michela Crisci, Francesco Crisci
Leukocytes are still difficultly highlighted inside platelets
aggregates, however, the boundary line between RBCs and
platelet aggregates/leukocytes is very clear. The boundary line
between RBCs, leukocytes and platelet aggregates is not
distinguishable.
PRF clot observation through SEM at low magnification (15x)
demonstrated that the clot shows a central concavity, which is a
fixation artefact. In the red portion of PRF clot, clots display
RBCs inside the fibrin mesh. RBCs are normal, but the fibrin
mesh is premature. At the boundary between the red and yellow
parts of the clot (buffy coat area), the SEM examination showed
leukocytes, appearing as spherical structures with an irregular
surface. Of these, the major part is small (m diameter) and
hence they could be essentially lymphocytes. Platelet aggregates
are evident along fibrin filaments. Over the “buffy coat” area, we
distinguish two different areas: the first area is made of thick
fibrin filaments and few sparse RBCs, possible due to
contamination; fibrin seems to be mature; the second area is made
of that strand of condensed platelets, observed at the optic
microscope, characterized by platelet and fibrin in dense, big
masses, due to ample aggregation and coagulation processes. This
aggregate is made of a thick, solid meshwork, and platelets seem
to be highly activated during the PRF preparation protocol.
At higher magnifications, fibrin is clearly organized in parallel
fibers, very thick and dense. In this organization, it is impossible
to distinguish the contained cellular elements.
The highest platelet and leukocyte density were found in
the first millimeter of the yellow clot, at the border with the red
clot. Platelet and leukocyte distribution become lower as we move
from the clot edge, and we didn’t found any platelet or leukocyte
beyond the first half of the yellow clot. Within the first 2 mm over
the boundary between the red and yellow clots, platelets and
leukocytes distribution is homogeneous enough over the whole
length of the clot. Over this boundary, as we move further from
the yellow/red boundary, more and more platelets (and
leukocytes) are grouped in specific areas, or central or centrifugal
platelet concentration. These zones show a high platelet/leukocyte
density, inside a cell-free matrix.
This type of architecture is similar in all clots,
independently of the patient, test tubes and compression method.
Platelet count showed clearly that there is almost no platelet inside
the RBCs layer, in PPP and in the exudate after the PRF clot
compression. Therefore, the major part of platelets coming from
the whole blood sample is gathered inside PRF membranes.
Leukocyte count confirmed that more than half of the leukocytes
is trapped inside the PRF membrane, and small lymphocytes seem
to be attracted in a selective manner, as confirmed by SEM
observations (Tabb.1, 2). These leukocytes do not seem to be
damaged during PRF preparation. This result has a huge clinical
impact as the leukocyte numbers implanted inside the membrane
is substantial, and small lymphocytes are particularly efficient in
inflammatory reactions’ regulation. Furthermore, thanks to L-PRF
cell composition, this biomaterial must be handled carefully, to
maintain the cellular content viable and stable.
Therefore, the most useful portion, under a surgical point
of view, is the whitish intermediate layer. Hence, it is necessary to
preserve a small RBCs layer at the PRF clot boundary, that
contains the bulk of platelets and leukocytes. The procedure must
be carried out with scissors, and it is operator-dependent, requiring
an accurate PRF architecture knowledge. Light compression of the
fibrin matrix makes it so that fibrin filaments are condensed and
they stick to each other. When PRF membranes are used in
surgical procedures, their resorption is slow, and it helps the fibrin
matrix remodeling to scar tissue.

Table 2. Count of erythrocytes, plateles and WBC on L-PRF membranes derived from clots at 0’ compared with those derived from clots at 60’ with
significance tests ( from Crisci et al. 2017) [12].
Type Membrane 0 min Membrane 60 min Between Membrane L-PRF 0-60 min
No./mL % No./mL % t-test* χ2
RBC 21,6012 0.0028% 193,966 0.0025% P=0.000 S P=0.266 NA
WBC 5,111.15 99.24% 5,036.86 97,80% P=0.007 NS P=0.993 NS
PLT 105,801 99.00% 97153 91.00% P=0.002 S P=1.000 NS
2 processing performed on two comparisons. Hypothetical content of RBC, WBC, PLT in the L-PRF membranes at 0 and 60 min with significance
tests. P> 0.05 =+0.5% no significant difference; P <0.01 = -1% significant difference.

Table 3. Leukocyte formula stabilized in whole blood (control group) and red clot after PRF membrane collecting (test group) (by Crisci et al. 2015).

Whole blood (%) Group 1 (%) Group 2 (%)

Cell type Mean Range Mean Range Mean Range
Neutrophils 51.8 49.7-53.2 72.1 66.1-77.1 66.4 60.9-71.4

Eosinophils 2.9 2.3-3.1 6.1 3.4-8.8 5.1 3.9-6.1

Basophils 0.5 0.3-0.8 0.1 0.0-0.3 0.4 0.1-0.9

Linfocytes 37.7 35.1-39.2 17.5 15.0-20.4 24.8 21.4-28.0

Monocytes 71.1 6.8-7.6 4.2 1.1-7.6 3.3 2.5-5.0

Total (Mean)/µl 6.900 3.500 3.600

(100%) (100%) (100%)

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 Leukocyte Platelet-Rich Fibrin (L-PRF), a new biomembrane useful in tissue repair: basic science and literature review
3. CONCLUSIONS
Concluding this essay, we can affirm that, to achieve a
standard procedure for PRF preparation as graft material for tissue
regeneration purposes, we suggest the employment of PRF
membrane’s region with the highest possible platelet enrichment
and, moreover, we suggest to avoid squeezing all of the PRF clot
plasma. Hence, it is advisable to compress the clot with a
compression device (L-PRF Wound Box). It’s difficult, therefore,
to control precisely the human-derived materials’ quality, like PRF
preparations, but is it important to apply the highest possible
quality-control check on PRF preparations before their clinical
application.
Currently, their delivery relies on poorly controlled bulk
release. As consequence, prolonged treatments require multiple
treatments e.g. numerous injections. This results in strongly
fluctuating growth factor concentrations, which impairs clinical
predictability. Biomaterials can act as controlled release devices,
which will allow for sustained or even on-demand delivery of
these growth factor cocktails. In addition, it can be envisioned that
biomaterials can covalently bind specific growth factors to locally
retain high levels of these molecules.
4. REFERENCES
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© 2018 by the authors. This article is an open access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
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Further clinical, histological and statistical studies are
required to understand the benefits of this new platelet
concentration technique. However, we cannot sweep aside the fact
that, when obtained from an autologous blood sample, produced
PRF is scarce and only a limited volume can be used. This is a
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information on its biology, efficacy and limits, to optimize its
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Cell migration plays a crucial role in the healing process.
MSCs represent a cell pool, able to reconstruct the damaged
tissue, and
endothelial cells contribute to angiogenesis. Migration models
induced by the supernatant of platelet concentrates’ culture do not
differ between the two types of cells.
The stronger MSCs and HUVECs migration were observed
as a reply to L-PRF. All of the above signifies that L-PRF could
be useful as a healing biomaterial, and as a natural anti-
hemorrhagic agent to be used at surgical sites.
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